Coactive Combinations of Antimicrobials with Dispersinb

This application is a continuation of U.S. application Ser. No. 17/908,832, filed Sep. 1, 2022, which is the 371 Application of PCT/CA2021/050269, filed Mar. 2, 2021, which claims the benefit of U.S. Provisional Patent Application No. 62/984,065, filed Mar. 2, 2020, which is incorporated by reference herein, the entireties of all of which are hereby incorporated by reference in their entireties.

The present invention relates to coactive DispersinB (DspB) formulations with antimicrobials and use of those formulations.

DspB is an enzyme that is naturally produced by a periodontal disease-associated oral bacterium,It specifically hydrolyses the glycosidic linkages of poly-beta 1, 6 N-acetylglucosamine (PNAG) leading to destabilization of biofilm structure and exposing biofilm-embedded bacteria. Purified recombinant DspB is shown to be active against diverse mammalian pathogens. In particular, PNAG is produced by a wide range of bacteria and fungi and is a key component in biofilm formation.

DspB cleaves PNAG, inhibiting bacterial adhesion and disperses the biofilm. This is especially useful for treating wounds and otic infections, which can become chronic due to the persistent nature of the bacterial biofilms. Once the biofilm is dispersed, the bacteria therein can be eradicated, and the infection can be remedied.

Bacteria is often eradicated or suppressed with antimicrobials or antibiotics.

However, the majority of broad-spectrum antimicrobials or disinfectants are surface active molecules that bind to and denature or inactivate proteins. For example:

As a result, when these antimicrobials are formulated with DspB, the DspB enzyme is typically inactivated before it can disperse the biofilm and release the bacteria. DspB is a protein/enzyme molecule that is susceptible to being inactivated by antimicrobial agents.

Previously, antimicrobial agents tested with DspB haven't been shown to work synergistically with DspB in a combination treatment or sequential treatment. In fact, the antimicrobial can be expected to inactivate the DspB.

It is a major challenge, therefore, to develop a formulation that enables DspB to function for a sufficient period of time to degrade PNAG based biofilms before a co-administered antimicrobial inactivates the DspB.

In embodiments, the present invention provides uses of an antimicrobial in a composition with DspB to enhance biofilm dispersal and inactivation of biofilm embedded bacteria, the antimicrobial comprising one or more of polyhexamethylene biguanide, polyaminopropyl biguanide, alexidine dihydrochloride, carvacrol, thymol, cinnamaldehyde, chloroxylenol, octenidine dihydrochloride, benzalkonium chloride, cetylpyridinium chloride, providone iodine, neomycin sulphate, gentamicin sulfate, polymyxin B sulfate, metronidazole, mupirocin, gramicidin, rifampin, rifabutin, rifapentine, or vancomycin.

In other embodiments, the present invention provides compositions comprising: DspB and an antimicrobial for enhancing biofilm dispersal and inactivation of biofilm embedded bacteria, the antimicrobial comprising polyhexamethylene biguanide, polyaminopropyl biguanide, alexidine dihydrochloride, carvacrol, thymol, cinnamaldehyde, chloroxylenol, octenidine dihydrochloride, benzalkonium chloride, cetylpyridinium chloride, providone iodine, neomycin sulphate, gentamicin sulfate, polymyxin B sulfate, metronidazole, mupirocin, gramicidin, rifampin, rifabutin, rifapentine, Manuka honey or vancomycin.

The invention contemplates the usefulness of families of antimicrobials and antibiotics with DspB, wherein the utility of some members of the class has been demonstrated. Such classes of antimicrobials and antibiotics are set out in Table 4, below.

The present invention encompasses any and all combinations of any of the polyols, polymers, salts, preservatives, antimicrobials, and buffers described herein, for stabilization of DspB.

The inventors have found that particular broad spectrum antimicrobials can be formulated with DspB without its inactivation for a sufficient period of time to enhance biofilm dispersal as well as inactivation of biofilm embedded bacteria. These formulations may be used for enhancing the activity of antimicrobials against biofilm embedded bacteria in chronic infections such as wounds, hard surfaces or in food processing or any other area where biofilm embedded bacteria contributing to human or animal infections.

Experiments were conducted against Gram− () and Gram+ () bacteria to determine whether DspB can maintain its enzymatic activity when mixed with a broad spectrum antimicrobial. In addition to determining if the antimicrobials and enzyme were active when mixed together, tests were also conducted to determine whether how long DspB maintained its activity. Tests were conducted both in a liquid buffer system and a gel formulation to assess the activity of antimicrobial and enzyme combinations.

orbiofilm were each grown in 96-well plate and washed three times to remove planktonic cells. Then biofilm was treated using 50 mM phosphate or citrate buffer, (no treatment control), buffer solutions containing DspB at 10-200 μg/ml, buffer solution containing antimicrobial alone, and buffer solution DspB plus antimicrobial for varying exposure times (10 min-3 h). After treatments, biofilm embedded bacteria was dispersed, diluted serially and plated on Tryptic soy agar plates. Data was analyzed using ANOVA and Tukey's test. Mean values were considered significantly different when P value was ≤0.05.

biofilm was grown in 96-well plate and washed three times to remove planktonic cells. Then biofilm was treated using different DspB and antimicrobial gel formulations for appropriate treatment duration (10 min-24 h). After treatment, gel was removed and washed 3 times using sterile water to remove excess gel. Then biofilm embedded bacteria was dispersed, diluted serially and plated on Tryptic soy agar plates.

biofilm was grown in 96-well plate and washed three times using sterile water to remove planktonic cells. Antimicrobials or antibiotics and DspB formulations were prepared in 50 mM phosphate or citrate buffer and stored at 37° C. for 2 h and used for treating biofilm at 0 and 2 h. Biofilm was treated using antimicrobials or antibiotics and DspB formulations for 10 min. After treatments, solutions were removed and washed 3 times to remove dispersed biofilm. Remaining biofilms in 96-well plates were stained using Crystal violet, unbound crystal violet was removed by washing the wells with sterile water, the biofilm bound crystal violet was eluted using 33% acetic acid, and the absorbance at 620 nm was taken using a microwell plate reader. Percent biofilm removal was calculated. Data was analyzed using ANOVA and Tukey's test. Mean values were considered significantly different when P value was ≤0.05.

DspB (200 μg/mL) and antimicrobial agent/antibiotic stocks were prepared in respective buffers. 10 mM paranitrophenol (PNP) in 400 mM sodium carbonate, and the buffer (same DspB stock) was used as the standard solution, and negative control, respectively. DspB stock and antimicrobial stock were mixed in equal proportions, and then 4×5 μl samples were taken at defined time points (0, 5, 10, 20, 30, 60, and 120 min) and used to measure the DspB enzyme activity. DspB enzymatic activity was measured using β-N-Acetylglucosaminidase assay kit from Sigma (product code CS0780) in 96-well microtiter plate following the manufacturer's instructions. The plate was incubated for 30 minutes before terminating the reaction. Data analysis was done in Excel. Student T test (paired) was performed for each time point separately using DspB activity in mixture and in control sample as two set of variables.

biofilm was grown in 96-well plate and washed three times to remove planktonic cells. Then biofilm was treated using different DspB formulations appropriate treatment duration (10 min-1 h). After treatments, gels were removed and washed 3 times with sterile distilled water to remove excess gel and dispersed biofilm. Remaining biofilms in 96-well plates were stained using Crystal violet and unbound crystal violet was removed by washing the wells with sterile water, the biofilm bound crystal violet was eluted using 33% acetic acid, and the absorbance at 620 nm was taken using a microwell plate reader.

DspB gel containing 200 μg/ml DspB, 16% Poloxamer 405 (PF127), 50 mM Phosphate buffer or citrate, 100 mM NaCl, pH 5.9, and 2× concentrated antimicrobial agent/antibiotics in 16% Poloxamer 405, 50 mM Phosphate buffer, 100 mM NaCl, pH 5.9 were mixed in equal proportions. 4×5 μL aliquots were withdrawn at 0, 5, 10, 20, 30, 60, and 120 min for measurement of DspB enzymatic activity. DspB enzymatic activity was measured using β-N-acetylglucosaminidase assay kit from Sigma (product code CS0780) in 96-well microtiter plate following the manufacturer's instructions. The plate was incubated for 30 minutes before terminating the reaction. Data analysis was done in Excel. Student T test (paired) was performed for each time point separately using DspB activity in mixture and in control sample as two set of variables.

The antimicrobial classes tested include: biguanides, monoterpenes/plant extracts or essential oil components, halophenols, phenylpropanoid, cationic surfactants, quaternary ammonium compounds, iodine compounds. Examples of specific biguanides tested include polyhexamethylene biguanide (PHMB), polyaminopropyl biguanide (PAPB), and alexidine. Examples of specific monoterpenes/plant extracts or essential oil components tested include carvacrol and thymol. An example of a halophenols tested includes chloroxylenol. An example of a phenylpropanoid tested includes cinnamaldehyde. An example of cationic surfactants tested includes octenidine dihydrochloride. Examples for quaternary ammonium compounds are cetylpyridinium chloride and benzalkonium chloride. An example of iodine compound is providone iodine.

Antibiotics that are commonly used for wound care application, and tested herein, include mupirocin, neomycin sulphate, gentamicin sulfate, polymyxin B sulfate, gramicidin, metronidazole, rifampin, rifapentine and vancomycin.

Tests were conducted to evaluate a) enhancement of inactivation of biofilm embedded bacteria by antimicrobial agents in the presence of DspB in buffer or gel (16% Poloxamer 405, 50 mM Phosphate/citrate buffer, 100 mM NaCl, pH 5.9) formulation (Method 1 and Method 2, respectively), and b) stability of DspB in the presence of antimicrobial agents in buffer or gel formulation were evaluated using biofilm dispersal assay (Method 3 and Method 5) and using enzymatic assay (Method 4 and 6), respectively.

Initial testing done with DspB and PHMB in buffer system showed that 10 μg/ml () and 20 μg/ml yielded similar inactivation of biofilm embedded bacteria and 10 min treatment was sufficient to disperse biofilms. When DspB was added to gel, colloidal matrix may delay diffusion and biofilm dispersal activity of DspB. Thus, for both buffer and gel formulations, DspB concentration was fixed at 20 μg/ml for most cases.

Some antimicrobials like octenidine dihydrochloride imparted negative effect on DspB when tested at 20 μg/ml. When 20 μg/ml DspB was mixed with this antimicrobial and biofilms were treated for 10 min, no biofilm dispersal was observed (data not presented). Thus, concentration of DspB was increased to 5× or 10× for further testing. DspB at 100-200 enhanced activity of octenidine dihydrochloride ().

DspB at 20 μg/ml did not enhance activity of berberine hydrochloride (). Rather, inactivation of biofilm embedded bacteria by berberine hydrochloride and 20 μg/ml DspB treatment was lower than berberine hydrochloride treatment alone (antagonism). Cinnamic acid treatment with DspB at 20 μg/ml did not show any enhancement of bacterial inactivation in biofilm (). Thus, further testing was not conducted with these compounds.

A summary of the antimicrobials or antibiotics tested with DspB in buffer formulations is set out in Table 1, and Examples 1 to 16. A summary of the antimicrobials or antibiotics tested with DspB in gel formulations is set out in Table 2 and Examples 17 to 35.

Based on the experimental results described herein, it is shown that at least polyhexamethylene biguanide, polyaminopropyl biguanide, alexidine dihydrochloride, carvacrol, thymol, cinnamaldehyde, chloroxylenol, octenidine dihydrochloride, benzalkonium chloride, cetylpyridinium chloride, providone iodine, neomycin sulphate, gentamicin sulfate, polymyxin B sulfate, gramicidin, metronidazole mupirocin, refampin, refapentine, vancomycin, Manuka honey and gramicidin are useful in allowing DspB to maintain its enzymatic activity for a reasonable amount of time when mixed. Thus, it was found that these antimicrobials and antibiotics in combination with DspB are useful in enhancing biofilm dispersal and enhancing inactivation of bacteria embedded therein.

Polyhexamethylene biguanide (PHMB), also referred to as polyhexanide, is a polymer typically used as a disinfectant and antiseptic. The chemical structure of PHMB is:

The present invention provides uses of PHMB with DspB, and compositions thereof, to enhance biofilm dispersal and inactivation of biofilm embedded bacteria. The PHMB with DspB may be applied in a liquid buffer formulation, a gel formulation, or in a manner known in the art.

In an embodiment, the concentration of PHMB is up to 0.15%. In a preferred embodiment, the concentration of PHMB is between 0.00313 and 0.1%. In a further preferred embodiment, when the composition is a gel formulation, the concentration of PHMB is about 0.1%.

In another embodiment, the concentration of DspB is between 10 and 20 μg/ml. In a preferred embodiment, the concentration of DspB is about 20 μg/ml.

Inactivation of biofilm embeddedandwere tested using Method 1. When PHMB was added with DspB, it enhanced (P<0.05) activity of PHMB against both(see) and(see).

is bar graph showing the increased susceptibility of biofilm-embeddedtreated with no antimicrobials in 50 mM Phosphate buffer (buffer), DspB at 10 μg/ml, PHMB at 0.0125, 0.05, and 0.1%, DspB+PHMB at 0.0125%, DspB+PHMB 0.05% and DspB and PHMB at 0.1% for 10 min at 37° C. Horizontal line was the detection limit of viable numbers. *Indicate significant reduction (P≤0.05) in viable numbers compared to buffer or DspB control. **indicate significant (P≤0.05) reduction in viable count compared to corresponding PHMB or DspB treatment alone. Note that DspB does not kill bacteria but instead dissolves the PNAG based biofilm that they reside in.

is bar graph showing the increased susceptibility of biofilm-embeddedtreated with no antimicrobials in 50 mM phosphate buffer (buffer), DspB at 20 μg/ml PHMB at 0.0125, 0.05, and 0.1%, +PHMB at 0.0125%, DspB+PHMB 0.05% and DspB and PHMB at 0.1% for 10 min at 37° C. Horizontal line was the detection limit of viable numbers. *Indicate significant reduction (P≤0.05) in viable numbers compared to buffer or DspB control. **indicate significant (P≤0.05) reduction in viable count compared to corresponding PHMB or treatment alone.

Compatibility between DspB and PHMB was tested using biofilm dispersal assay (Method 3) and enzymatic assay (Method 4). Results showed that ≥50% biofilm dispersal was observed when DspB and PHMB were added together (see). This result and the enzymatic assay result (see) suggest that DspB retained its activity for up to 2 h in the presence of PHMB.

is a bar graph showing biofilm dispersal activity of 50 mM phosphate buffer (buffer), PHMB 0.1% alone, 20 μg/ml DspB alone and 20 μg/ml DspB plus 20 μg/ml polyhexamethylene biguanide (PHMB). Results showed that DspB retained its activity for up to 2 h in the presence of PHMB. *Indicate significant reduction (P≤0.05) in biofilm dispersal compared to DspB control.

is a bar graph showing activity of DspB in the presence of 0.1% PHMB. DspB concentration used in mixture (DspB+PHMB) was 100 μg/mL in 50 mM phosphate buffer and it's % activity was calculated compared to the activity of 100 μg/mL DspB (considered 100% activity) as a control sample. Results showed that DspB was active for up to 2 h. *Indicate significant reduction (P≤0.05) in DspB activity compared to DspB control.

Polyaminopropyl Biguanide (PAPB) is commonly used as a disinfectant and a preservative. The chemical structure of PAPB is:

The present invention provides uses of PAPB with DspB, and compositions thereof, to enhance biofilm dispersal and inactivation of biofilm embedded bacteria. The PAPB with DspB may be applied in a liquid buffer formulation, a gel formulation, or in a manner known in the art.

In an embodiment, the concentration of PAPB is up to 0.25%. In a preferred embodiment, the concentration of PHMB is between 0.05 and 0.2%. In a further preferred embodiment, when the composition is a gel formulation, the concentration of PHMB is about 0.1%.

In another embodiment, the concentration of DspB is between 10 and 20 μg/ml. In a preferred embodiment, the concentration of DspB is about 20 μg/ml.

Inactivation of biofilm embedded S. epidermidis was tested using Method 1. When PAPB was added with DspB, DspB enhanced (P<0.05) activity of PAPB against(see).

is bar graph showing the increased susceptibility of biofilm-embeddedtreated with no antimicrobials in 50 mM citrate buffer (buffer), DspB at 20 μg/ml, PAPB at 0.05, 0.1, and 0.2%, DspB+PAPB at 0.05%, DspB+PAPB 0.1% and DspB and PAPB at 0.2% for 10 in at 37° C. Horizontal line was the detection limit of viable numbers. *Indicate significant reduction (P≤0.05) in viable numbers compared to buffer or DspB control. **indicate significant (P≤0.05) reduction in viable count compared to corresponding PAPB or DspB treatment alone.

Alexidine dihydrochloride (alexidine) is a biguanide class antimicrobial with a chemical structure of:

The present invention provides uses of alexidine with DspB, and compositions thereof, to enhance biofilm dispersal and inactivation of biofilm embedded bacteria. The alexidine with DspB may be applied in a liquid buffer formulation, a gel formulation or in a manner known in the art.